The long term objective of this research proposal is to define the mechanism by which the rat liver ATPase is synthesized, transported and assembled into the functional unit (H ion-translocating ATPase) on the inner face (M side) of the inner mitochondrial membrane. It is important to understand this process because it provides a model of intracellular protein transport which will provide a basis for understanding the defective assembly of the ATPase in Zajdela hepatoma. A method has been developed to selectively disaggregate the H ion-translocating ATPase which allows purification of the ATPase still associated with part of its binding site. The Type I ATPase contains a 26.5 kilodalton protein not found in F1-ATPase. It is proposed that the 26.5 kilodalton binding protein is composed of the oligomycin sensitivity conferral protein and FC2(F6). Using the Type I ATPase as a tool, we will determine what subunit(s) of F1 and Fo are involved in the binding of the ATPase to the membrane. The extent of identity of the 26.5 kilodalton protein to OSCP, FC2, sigma and epsilon subunit of F1 will be determined. Reconstruction of the ATPase binding site will be accomplished with the purified 26.5 kilodalton binding protein, the portion of Fo involved in the binding of the 26.5 kilodalton protein, liposomes and purified ATPase. Specific ATPase binding will be distinguished from non-specific ATPase binding by the KD and extent of binding. This reconstructed unit will constitute the minimal structural unit for the ATPase binding site.